Embryonic stem cells have great potential in therapeutic use to replace diseased or damaged tissues because they have the unique capability of giving rise to any cell type of the body while perpetuating their own identity, even after repeated cell divisions. Recent advances in this area have resulted in a new way to generate stem cells from specialized adult cells by introducing 3 to 4 genes encoding proteins called stem cell factors, which are highly active in natural stem cells, into these adult cells using viruses as the carrier. These derivatives are called induced pluripotent stem (iPS) cells and have properties that are very similar to those of embryonic stem cells. Because iPS cells can be generated from the patient’s own tissues, problems associated with immune rejection are avoided. Furthermore, this process does not use embryos, so there are no ethical concerns. Unfortunately, the use of viruses to generate these cells is problematic because the virus may also activate harmful genes in the cells, such as those that cause cancer. We recently developed a way to switch on inactive genes in human cells using small RNA molecules instead of viruses, and coined the technique ”RNAa” for RNA-induced gene activation. We have shown that RNAa can induce robust and prolonged activation of a variety of genes. RNAa therefore seems well suited to replace virus-mediated reprogramming as a means to generate iPS cells. The main goal of this application is to develop a novel method of transforming adult cells into stem cells without using viruses. Accomplishment of our study will bring iPS cells one step closer to the clinical application of stem cell therapy.

Statement of Benefit to California:

The aim of this application is to develop new approaches for the generation of pluripotent stem cell lines without using virus as the gene expression vector. Stem cells so generated can be used to replace diseased or damaged tissues without the concern of virus-related adverse effects such as insertional mutagenesis. Success of these approaches will benefit the health of the population and the economy of the State of California. Californians suffer many diseases and injuries that are treatable by using stem cells, such as Parkinson’s and Alzheimer’s disease, diabetes and cancer. The new stem cell lines and reagents we generate will likely be commercialized by California-based biotechnology companies and thus generate revenue and new job opportunities for the state.

Progress Report:

We proposed to generate human induced pluripotent stem (iPS) cells using a virus-free technique called RNA activation (RNAa). RNAa is a newly identified gene regulation mechanism by which promoter-targeting double-stranded small RNA also known as small activating RNA (saRNA) can induce gene expression. Our approach to iPS cell derivation is through simultaneous activation of stem cell factors including OCT4, NANOG, SOX2 and KLF4 using RNAa. Currently we are focusing on Aim 1 to screen saRNAs that can activate individual stem cell factors as specified in our proposal. To facilitate and expedite such screening process, besides the screening method we originally proposed, that is through expression analysis of endogenous genes, we have adapted three new strategies. They include the use of a lentiviral vector based reporter system, use of a mouse retroviral iPS reprogramming platform and use of established cell lines. We successfully established reporter systems for all four human stem cell factors including OOT4, NANOG, SOX2 and MYC. The reporter systems allow for high throughput screen of saRNAs in primary fibroblast cells. Using a reporter vector for MYC promoter, we identified several activating saRNAs for MYC gene in human fibroblast BJ cells. We are currently using the same system to screen saRNAs for other genes. We are also using the mouse iPS reprogramming platform to screen saRNAs that help reprogramming without first knowing their ability in gene activation and have successfully identified four saRNAs for mouse Myc gene. Replacing Myc retrovirus by the saRNAs significantly increased the number of iPS clones compared to the number of clones from cells infected by the remaining 3 factors (Oct4, Sox2 and Klf4) alone. This result provided proof of principle that saRNAs can be directly screened on an iPS reprogramming platform. We will next move this screening method to human cells and to achieve iPS reprogramming by replacing virus for other stem cell factors particularly OCT4. Since established cancer cell lines are easy to transfect with small RNA, we also used human cancer cell lines to screen saRNAs for KLF4 gene and were able to identify one potent saRNA. RNAa-mediated KLF4 activation caused the modulation of its regulated genes including several cell cycle genes. These results were fully recapitulated by retroviral vector based overexpression of KLF4, thus validating RNAa as a method of restoring endogenous gene function. Ongoing research is identifying saRNAs for additional stem cell factors and maximizing gene activation by optimizing saRNA nucleofection and by combinatorial treatment with epigenetic modifying agents.

We proposed to generate human induced pluripotent stem (iPS) cells using a virus-free technique called RNA activation (RNAa). RNAa is a newly identified gene regulation mechanism by which promoter-targeting double-stranded small RNA also known as small activating RNA (saRNA) can induce gene expression. Our approach to iPS cell derivation is through simultaneous activation of stem cell factors including OCT4, NANOG, SOX2 and KLF4 using RNAa. Currently we are focusing on Aim 1 and 2 of this project to screen saRNAs that can activate individual stem cell factors and to use the identified saRNAs to replace one viral factor at a time to reprogram iPS cells from somatic cells. We have identified saRNAs for OCT4, KLF4 and NANOG. Replacing OCT4 virus in the OSKM four factor reprogramming recipe (OCT4, SOX2, KLF4 and MYC) with an OCT4 saRNA led to the derivation of iPS-like colonies from adipose tissue derived stem cells (ADSCs). These results provided proof of principle that saRNAs can be used for iPS reprogramming. Ongoing research is identifying saRNAs for additional stem cell factors and maximizing gene activation by optimizing iPS reprogramming protocol. Eventually all viruses will be replaced with their corresponding saRNAs to generate virus-free iPS cells.

We proposed to generate human induced pluripotent stem (iPS) cells using a virus-free technique called RNA activation (RNAa). RNAa is a newly identified gene regulation mechanism by which promoter-targeting double-stranded small RNA also known as small activating RNA (saRNA) can induce gene expression. Our approach to iPS cell derivation is through simultaneous activation of stem cell factors including OCT4, NANOG, SOX2, KLF4 and C-MYC using RNAa. Currently we are focusing on Aim 1 and 2 of this project to screen saRNAs that can activate individual stem cell factors and to use the identified saRNAs to replace one viral factor at a time to reprogram iPS cells from somatic cells. We have identified saRNAs for OCT4, KLF4, NANOG and C-MYC. Replacing OCT4 virus in the OSKM four factor reprogramming recipe (OCT4, SOX2, KLF4 and MYC) with an OCT4 saRNA led to the derivation of iPS-like colonies from adipose tissue derived stem cells (ADSCs). These results provided proof of principle that saRNAs can be used for iPS reprogramming. Ongoing research is maximizing gene activation and iPS induction by tweaking saRNA design, use of small molecule compounds and optimizing iPS reprogramming protocols. Eventually all viruses will be replaced with their corresponding saRNAs to generate virus-free iPS cells.

We proposed to generate human induced pluripotent stem (iPS) cells using a virus-free technique called RNA activation (RNAa). RNAa is a newly identified gene regulation mechanism by which targeting gene regulatory elements known as promoters using small activating RNA (saRNA) can induce gene activity. Our strategy of iPS cell derivation relies on the use of saRNAs to stimulate the endogenous expression of several stem cell factors including OCT4, NANOG, SOX2, KLF4 and C-MYC which are known to be able to induce iPS cells if they are forced to express using viral vectors. We took a three-step approach involving first screening saRNAs that could activate individual stem cell factors, then using the identified saRNAs to replace one viral factor at a time, and eventfully replace all viral factors to reprogram iPS cells from somatic cells. We have identified saRNAs for OCT4, KLF4, NANOG and C-MYC. Replacing OCT4 virus in the OSKM four factor reprogramming recipe (OCT4, SOX2, KLF4 and MYC) with an OCT4 saRNA led to the derivation of iPS-like colonies from adipose tissue derived stem cells (ADSCs). We also found that a NANOG saRNA which induces NANOG expression can antagonize retinoic acid-induced differentiation of stem cells. These results provided proof of principle that saRNAs can be used for cell fate manipulation. Despite promising results have been achieved, significant obstacles need to be circumvented before we reach our final goal of deriving virus-free iPS cells. Ongoing research is optimizing saRNA-induced reprogramming by tweaking saRNA design and iPS reprogramming protocols and use of small molecule compounds. Alternative strategy of direct conversion of one cell type to another by RNAa is also being tested.